Recent studies of nematodes and fruit flies have revealed that mutations in the genetic pathways controlling anti-oxidant defense systems can significantly alter lifespan. Similar studies have also revealed that genes thought to participate in the mechanisms of aging are also important for proper development. These issues in mammals are poorly understood. Conventional gene knockout approaches are extremely useful for investigating developmentally important genes in mice. However, as homozygous null knockout mice often survive only to embryonic or postnatal stages, conventional strategies provide only limited information about the role of developmentally important genes in the aging process. Since death is such an irreversible event, processes affecting aging in adults can not be studied. One such developmentally essential gene encodes manganese superoxide dismutase (MnSOD), a mitochondrial anti-oxidant enzyme. Conventional gene-targeting strategies produce homozygous null MnSOD-deficient mice that die during postnatal development. We have designed a novel strategy for conditionally rescuing such embryonic lethal phenotypes. Our prototype replacement vector for targeting the gene encoding MnSOD has three key features. First, it deletes essential mouse MnSOD catalytic domains. Second, it introduces a catalytically active human MnSOD that is under the control of the endogenous mouse Sod2 promoter. Thus, human MnSOD will replace the defective mouse MnSOD and functionally rescue the developmental lethal phenotype in homozygous null mice. The third key feature is that human MnSOD expression can be suppressed by administration of a common antibiotic at any stage of development. This approach will provide, for the first time, adult mice with a complete deficiency in MnSOD, a key mitochondrial anti-oxidant defense enzyme. Our knock-out, knock-in Sod2 homozygous nulls will be valuable for investigating the role of oxidative stress on lifespan and age-related pathologies. Our conditional rescue gene-targeting strategy is applicable to any gene of interest and thus is likely to find widespread use in many areas of mammalian biology.